The study of protein structure and function has historically relied upon the chemical properties that are available using the R-groups of the naturally occurring amino acids. Unfortunately, every known organism, from bacteria to humans, encodes the same twenty common amino acids (with the rare exceptions of selenocysteine (see, e.g., Bock et al., (1991), Molecular Microbiology 5:515-20) and pyrrolysine (see, e.g., Srinivasan, et al., (2002), Science 296:1459-62). This limited selection of R-groups has restricted the study of protein structure and function, where the studies are confined by the chemical properties of the naturally occurring amino acids.
A general methodology has been developed for the in vivo site-specific incorporation of chemically diverse unnatural amino acids with novel physicochemical and biological properties into proteins in both prokaryotic and eukaryotic organisms (Wang et al., Science 292, 498-500 (2001); Chin, et al. Science 301, 964-967 (2003); Wang and Schultz, Angew. Chem. Int. Ed. 44, 34-66 (2005)). This method relies on a unique codon-tRNA pair and corresponding aminoacyl-tRNA synthetase (aaRS, or simply RS) for each unnatural amino acid that functions efficiently in protein translation, but do not cross-react with any of the endogenous tRNAs, RSs, amino acids or codons in the host organism (i.e., it must be orthogonal). The use of such orthogonal tRNA-RS pairs has made it possible to genetically encode a large number of structurally diverse amino acids including those with unique chemical (Wang et al., Proc. Natl. Sci. Acad. USA. 100, 56-61 (2003)) and photochemical reactivity (Chin et al., Proc. Natl. Acad. Sci. USA 99, 11020-11024 (2002); Wu et al., J. Am. Chem. Soc., 126, 14306-14307 (2004)) as well as glycosylated (Zhang et al., Science 303, 371-373 (2004)) fluorescent (Wang and Schultz, Angew. Chem. Int. Ed. 44:34-66 (2005)), metal binding (Wang and Schultz, Angew. Chem. Int. Ed. 44:34-66 (2005)) and redox active amino acids (Alfonta et al., J. Am. Chem. Soc. 125:14662-14663 (2003)). One particular mutant MjtRNA-Tyr(CUA)-MjTyrRS pair has been particularly useful for encoding new amino acids in E. coli (Wang and Schultz, Chem. Biol. 8:883-890 (2001)).
However, despite the success of this technique in incorporating a diverse array of unnatural amino acids in vivo, the efficiency of the expression system for the production of mutant proteins containing unnatural amino acids has not been optimized, and suppression efficiency of the orthogonal system to overcome the selector codon can be poor. There is a need in the art to develop reagents to improve the suppression efficiency of orthogonal translation systems. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.